Sialyl transferases are important enzymes for use in the preparation of pharmaceutical compounds and as diagnostic and clinical reagents for specifically reacting carbohydrates. All sialyl transferases (ST) catalyze the general reaction: EQU sialyl-CMP+acceptor.fwdarw.sialyl-acceptor+CMP
There are a large number of ST enzymes distinguished on the basis of the type of linkage formed (e.g., alpha-2,3 or alpha-2,6) and on the acceptors for which they are specific. It has been estimated that 10-12 specific STs are required to synthesize all of the sialylated structures in mammalian systems. (Paulson, J. C. et al. J Biol Chem (1987) 262:17735-17743.)
Purified forms of specific ST enzymes are useful both as research tools and as synthetic reagents for the manufacture of pharmaceuticals (Gross, H. J. et al. Eur J Biochem (1987) 168:595). For example, these enzymes have been used in the regiospecific and stereospecific synthesis of disialylated tetrasaccharides analogous to M and N blood group determinants of glycophorin A (DeHeij, H. T. et al. J Carbohydrate Chem (1988) 7:209-222). The necessity for purified enzymes in order to produce desired sialylated oligosaccharides was pointed out by, for example, David, et al. Pure Appl Chem (1987) 59:1501-1508.
Because of the recognized need for purified forms of the specific STs, various approaches have been made to purification of these enzymes from natural sources, especially those with the highest concentrations of STs, such as liver, platelets and erythrocytes. A general discussion of such purification procedures is found in Beyer, T. A. et al. Adv Enzymol (1981) 52:23-175, and the desirability of affinity purification is recognized. Most commonly used affinity ligands have been purine and pyrimidine nucleotides linked to solid supports through spacer arms (Barker, R. et al. Methods Enzymol (1974) 34:479-491. This type of affinity ligand is not only expensive, but has proved unsatisfactory in use.
Purification of various specific ST enzymes has been reported by a number of groups, as follows:
Joziasse, et al. J Biol Chem (1985) 260:4941 report the purification of alpha 2,3-ST from human placenta;
Heij, et al. J Carbohydrate Chem (1988) 7:209-222 have reported purification of alpha 2,6-ST from rat liver;
Weinstein, et al. J Biol Chem (1982) 257:13835-13844 report purification of alpha 2,3-ST which uses Gal(beta)1,3(4)GlcNAc as acceptor and/or alpha 2,6-ST which uses Gal(beta)1,4GlcNAc as acceptor, from rat liver;
Conradt, et al. "Sialic Acids" (1988) Proceedings of the Japanese-German Symposium, Berlin, p. 104 report purification of an alpha 2,3-ST from porcine liver which uses Gal(beta)1,3GalNAc as acceptor;
Sadler, et al. J Biol Chem (1979) 254:4434-4443; ibid 5934-5941, report purification of alpha 2,3-ST from porcine submaxilary glands, which uses beta-Gal as acceptor;
Higa, et al. J Biol Chem (1985) 260:8838-8849 report purification of alpha 2,6-ST from bovine submaxilary glands which uses GalNAc as acceptor; and
Paulson, et al. J Biol Chem (1977) 252:2356-2362 report purification of alpha 2,6-ST from bovine colostrum, which uses beta-Gal as acceptor.
Of the foregoing, the enzymes prepared by Weinstein et al. and by Sadler et al. were purified to homogeneity.
A variety of techniques were used in the foregoing preparations, most commonly an affinity step which involves CDP-hexanolamine Sepharose as an affinity support. The column equilibration and elution conditions required for isolation using this kind of generalized affinity support were very critical. Furthermore, the affinity support itself is difficult to synthesize.
Recently, Sticher, et al. Biochem J (1988) 253:577-580 described a simple 3-step procedure for purification of Gal(beta)1,4GlcNAc-specific alpha 2,6-ST from rat liver using dichromatography on Cibacron Blue F3GA and FPLC, to purify the enzyme to apparent homogeneity. However, the absence of accompanying alpha 2,3-ST activity was not convincingly established.
Putatively pure rat liver Gal(beta)1,4GlcNAc-specific alpha 2,6-ST and porcine submaxilary gland Gal(beta) 1,3GalNAc-specific alpha 2,3-ST are commercially available; alpha 2,3-ST specific for Gal(beta)1,3GlcNAc cannot, however, be prepared easily.
Thus, although various ST enzymes have been obtained by prior art methods, there is no simple procedure for isolating a desired ST of given specificity. No previously used method has taken advantage of acceptor specificity in the design of an affinity ligand.
One acceptor of use in the present invention is Gal(beta)1,4GlcNAc (LacNAc). Elices, et al. Arch Biochem Biophys (1987) 254:329-341 utilized LacNAc derivatized to the commercially available Synsorb.TM. support beads for assay of alpha 2,3-ST and alpha 2,6-ST from rat liver. However, these supports were not used for purification of the enzymes.